The ultimate goal of this research project is to develop novel biologically based, dental and craniofacial skeletal repair and regeneration therapies in humans. We hypothesize that improved knowledge of dental stem cell (DSC) properties, and characteristics, combined with improved knowledge of tissue engineering strategies to reliably generate mineralized dental and craniofacial tissues of predictable size and shape, will result in the development of novel and effective, clinically relevant therapies for humans. To address this hypothesis, four specific aims are proposed, all of which will exclusively use human DSCs harvested from extracted human teeth. First, we will characterize the properties of individually harvested human dental tissues, to establish the normal range of DSC properties, to correlate DSC properties with the age and overall health of the donor, and to correlate these properties with the ability to successfully use harvested dental stem cells for dental tissue regeneration therapies. Next, we will test four different scaffold materials, carefully chosen for their defined physical properties, for their ability to generate bioengineered human tooth crowns of predictable size and shape. Third, we will expand upon our results demonstrating that silk scaffolds can be used to generate osteodentin of predicted size and shape, to bioengineer full sized, functional human tooth root equivalents that can support a synthetic or bioengineered tooth crown. Silk scaffold fabrication methods, porosity, degradation rates, and added growth factor peptides, will be systematically evaluated. As recommended in prior review, Aims 2 and 3 will be performed in both subcutaneous and minipig mandibular implant models. Finally, in Aim 4, we will combine our tooth crown, root, and craniofacial skeletal regenerative strategies, again using the Yucatan mini pig mandibular implant model, to generate successive 1st, 2nd and 3rd generation replacement teeth. For each of the proposed aims, detailed developmental analyses of bioengineered dental implant tissues will be performed in order to better understand, and devise strategies to improve dental tissue regeneration. Our novel approach to tooth repair and regeneration, using human DSCs, and state of the art scaffold fabrication methods, combined with our extensive expertise in Developmental Biology and Tissue Engineering, have the potential to provide new and improved, biologically based repair and regeneration strategies, using autologous tissues. The successful accomplishment of the proposed studies would dramatically alter the field of dentistry as it currently exists, extending clinically relevant dental repair therapies to include biologically based dental materials with properties closely matching those of naturally formed dental tissues.